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Cell DensityApplications eBook
Hamilton's on-line cell density sensors have been used by a variety of customers for applications ranging from feed control to continuous processing. This eBook provides a summary of relevant data and references to published research that employed those sensors.
Publication Overview
IntroductionDiscover On-Line Cell Density............................................... 3
About Incyte ......................................................................... 4
About Dencytee .................................................................... 5
By ApplicationMonitoring Viable and Total Cells at the Same Time ............. 6
Feed Control ......................................................................... 7
Continuous Processing ......................................................... 8
Cells on Microcarriers ........................................................... 9
Correlation of On-Line to Off-Line Measurements ...............10
By Cell Line / StrainMammalian Cells (CHO) ........................................................ 6
Mammalian cells (BHK) ......................................................... 9
Insect cells (SF9) ..................................................................10
Yeast (Orgatae).....................................................................11
Bacteria (E. coli) ...................................................................12
Filamentous Fungi (Absidia)..................................................13
2
Discover On-Line Cell DensityInnovative Methods that Improve and Streamline Cell Density MonitoringTraditional cell density measurement and monitoring is time- and labor-intensive. It also lacks real-time, continuous data and makes assumptions based off of off-line sampling and manual cell counting.
Hamilton Process Analytics has revolutionized and modernized this process. Rather than counting the cells directly, Hamilton sensors use the principles of permittivity measurement (capacitance per area) and light absorbance which correlate to the cell count in solution at the production line. It’s an improvement that saves time and allows for advanced process control.
The following pages detail real applications of these sensors and measurement principles to show the benefits and opportunities offered by this shift in technology.
3
About IncyteReal-Time, On-Line Viable Cell Density Measurement by PermittivityIncyte sensors measure permittivity in the on-line process to give a continuous, in-situ reading of viable cells in solution. This eliminates time-intensive manual cell counting.
Incyte Highlights: ■ Not influenced by changes in the media, microcarriers, dead cells, or debris
■ Can increase yield and lower production costs
■ Can detect changes in cell physiology with frequency scanning
■ Offers precise data to control harvesting in continuous culture
■ Offers early detection of process deviations
■ Available in optimized versions for multiple conductivity ranges
PL ATINUM ELECTRODES
VIABLE CELLS POL ARIZE
ALTERNATING ELECTRIC
FIELDS
DE AD CELLS HAVE DAMAGED MEMBR ANES AND DO NOT POL ARIZE
4
About DencyteeReal-Time, On-Line Total Cell Density Measurement by Optical DensityDencytee sensors determine optical density by measuring how cells in solution absorb and scatter light. These on-line, continuous readings eliminate the need for a technician to manually sample and count cells off the production line.
Dencytee Highlights: ■ Simple on-line measurement of cell growth
■ Reliable values during the growth phase
■ Early detection of process deviations
LIGHT SOURCE INCRE ASES INTENSIT Y TO COMPENSATE
FOR THE TURBIDIT Y OF THE CULTURE BROTH
LIGHT DETECTOR
5 mm WINDOW
5
Return to Index
Mammalian Cells (CHO)
Viable and Total CellsObtaining relevant on-line data about viable and total cell density.
■ Incyte correlates well with viable cell counts throughout process
■ Incyte and Dencytee together can be used to estimate viability
Kandra, Kulwant, et al. On-Line Monitoring of CHO Cell Culture. Hamilton Company, 2014.
165
110
55
0
165
110
55
0
0 65 130 195 260
Time [h]
Via
ble
Cel
l Cou
nt [1
05 ce
lls/m
L]
Via
ble
Cel
l Cou
nt [1
05 ce
lls/m
L]
■ Viable Cell Counts ■ Dencytee Measurement ■ Incyte Measurement
6
Return to Index
Mammalian Cells (CHO)
Optimize the ProcessMonitoring permittivity provides the possibility to control feeding and enable optimal nutrition supply.
In this study, alanine is an indicator for the protein of interest. As glutamine nears depletion, the metabolism of the cells change and alanine is similarly depleted. At that point there is no productivity.
■ Drop in permittivity, as measured by Incyte, triggered glucose and glutamine feeding
■ Expanded alanine production phase of the protein of interest by three days as compared with a non feed-controlled process (greatly reducing workload of new batch startup)
Roche (2005) CHO Biomass nutrients limit. Poster at ESACT19
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6
5
4
3
2
1
0
11
10
9
8
7
6
5
4
3
2
1
0
0 50 100 150 200 250
Time [h]
Per
miti
vity
[pF/
cm]
Bio
volu
me
[Litr
e]
Glutamine [mmol/L] Alanine [mmol/L] Incyte MeasurementGlucose [g/L]
7
Return to Index
Mammalian Cells (CHO)
Continuous ProcessingViable Cell Density monitoring correlates with the off-line Cedex counts.
■ Incyte and Dencytee correlate throughout continuous (perfusion) process
■ No sensor adjustment needed when harvesting
Real-time Cell Density Measurement for PAT Applications, Dr. Heino Büntemeyer, Anica Schmidt, Zellkulturtechnik, Technische Fakultät, Universität Bielefeld.
Time [h]
VC
D [1
0^5
Cel
ls/m
L]
TCD
[OD
880 ]
6.30
5.30
4.30
3.30
2.30
1.30
140
120
100
80
60
40
20
0
0 50 100 150 200 250 300
■ Off-Line ■ Incyte Measurement ■ Dencytee Measurement
8
Return to Index
Mammalian Cells (BHK)
Cells on MicrocarriersThe correlation of permittivity to viable cell count is not influenced by the presence of microcarriers.
■ Incyte helps avoid misinformed decisions caused by outliers (marked with the red circle)
■ Microcarriers have no impact on permittivity measurements
On-ine Viable Cell Density Measurement on Microcarriers; Marlene Frank, Ruma Lüthi, Iris Poggendorf, Caspar Demuth, Waedenswil
Time [h]
Per
mitt
ivity
[pF/
cm]
Via
ble
Cel
l Cou
nt [c
ells
/mL]
7.0E+06
6.0E+06
5.0E+06
4.0E+06
3.0E+06
2.0E+06
1.0E+06
0.0E+06
FEED
140
120
100
80
60
40
20
0
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00
■ Viable Cell Count ■ Incyte Measurement
This data point is an outlier that can be seen
as compared to the Incyte measurement.
Without Incyte, an incorrect process estimation would
likely happen.
9
Return to Index
Insect Cells (SF9)
Biovolume to Permittivity
Permittivity shows a high correlation to off-line measurements of biovolume.
■ Incyte correlates with biovolume throughout process
Related work: On-Line Monitoring of Infected Sf-9 Insect Cell Cultures by Scanning Permittivity Measurements and Comparison with Off-Line Biovolume Measurements. Sven Ansorge. Cytotechnology (2007) 55:115–124
SF9 Insect Cell Growth and Infection Monitoring
Linear Relationship Permittivity - Biovolume
■ Biovolume ■ Biovolume
Permitivity [pF/cm]
Bio
volu
me
[µm
3 /m
L]
Bio
volu
me
[µm
3 /m
L]
10
9
8
7
6
5
4
3
2
1
0
10
9
8
7
6
5
4
3
2
1
00 1 2 3 4 5
y = 1.9788x
Permitivity [pF/cm]
Bio
volu
me
[µm
3 /m
L]
Bio
volu
me
[µm
3 /m
L]
10
9
8
7
6
5
4
3
2
1
0
10
9
8
7
6
5
4
3
2
1
00 1 2 3 4 5
y = 1.9788x
INFE
CTI
ON
Culture Time [h]
Per
miti
vity
[pF/
cm]
Bio
volu
me
[µm
3 /m
L]
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
10
9
8
7
6
5
4
3
2
1
00 20 40 60 80 100 120 140 160
Esteban, G. (2006) On-Line Viable Cell Density and Physiological States Monitoring by Dielectric Spectroscopy sf9 Growth and Infection Process. Poster at Cell Culture Engineering X.
■ Incyte Measurement ■ Incyte Measurement
10
Return to Index
Yeast (Orgatae)
Concentration to PermittivityViable cell density monitoring correlates with the off-line measurement.
■ Good correlation with dry weight throughout process
■ On-line monitoring provides data 24 hours/day and 7 days/week, as compared with off-line sampling that is only done when technicians are on duty
Xylitol Fermentation Experiments with Different Yeast Strains, Csaba Feher, Zsolt Barta, Budapest University of Technology and Economics
Propagation of Ogateae Yeast in Bench-top Bioreactor
12
10
8
6
4
2
0
2.50
2.00
1.50
1.00
0.50
0.00
0 50 100 150 200 250
Time [h]
Per
miti
vity
[pF/
cm]
Cel
l Con
cent
ratio
n [g
/L]
■ Off-Line Cell Count ■ Incyte Measurement
11
Return to Index
Bacteria (E. coli)
Dry Weight to Permittivity
Viable cell density monitoring correlates with the dry weight measurement.
■ Incyte correlates to dry cell weight throughout feed and differs after induction, when production phase starts
■ IPTG Induction starts the production phase and leads to increased bacterial size and permittivity
Bioprocess Biosyst Eng 2012_Horta et al_iIntensification of high cell-density cultivations of E. coli
140
120
100
80
60
40
20
0
140
120
100
80
60
40
20
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Time [h]
Cx
[g/L
]
Cx
[g/L
]
FEE
D S
TAR
TS
IPTG
IND
UC
TIO
N
■ Cell Dry Weight ■ Incyte Measurement
12
Return to Index
■ Cell Dry Weight ■ Incyte Measurement
INRA - French Agronomical Research Institute
Filamentous Fungi (Absidia)
Dry Weight to Permittivity
Viable cell density monitoring correlates with the off-line method.
■ Filamentous fungi show a high heterogeneity during cultivation, therefore reliable off-line sampling is very difficult
■ Incyte can be used for on-line measurement of viable microorganisms – no sampling needed
25
20
15
10
5
0
12
10
8
6
4
2
0
0 10 20 30 40 50
Time [h]
Per
mitt
ivity
[pF
/cm
], C
ondu
ctiv
ity [m
S/c
m]
Dry
Wei
ght [
g/L]
13
Talk to a Hamilton Expert About Your
Cell Density ApplicationNow that you’ve seen Incyte and Dencytee in action, contact a Hamilton sales representative about how to apply this technology to your application and process.
[email protected] US: 800-648-5950
Europe: +41-58-610-10-10
Hamilton Americas & Pacific RimHamilton Company Inc.4970 Energy WayReno, Nevada 89502 USATel: +1-775-858-3000Fax: [email protected]
Hamilton Europe, Asia & AfricaHamilton Bonaduz A.G.Via Crusch 8CH-7402 Bonaduz, SwitzerlandTel: +41-58-610-10-10Fax: [email protected]
To find a representative in your area, please visit hamiltoncompany.com/contacts.
Web: www.hamiltoncompany.comUSA: 800-648-5950Europe: +41-58-610-10-10
©2018 Hamilton Company. All rights reserved. All other trademarks are owned and/or registered by Hamilton Company in the U.S. and/or other countries.
Lit. No. L30019 — 01/2018
